It is becoming increasingly clear that G protein-coupled receptor (GPCR) kinases (GRKs) play a critical role in modulating myocardial signaling and function. GRKs phosphorylate agonist-occupied GPCRs triggering the process of desensitization, leading to the loss of down-stream signaling. GRK action on GPCRs promotes B-arrestin binding to receptors, which leads to receptor internalization and resensitization as well as activation of novel signaling cascades. As delineated during the first funding cycle of this PPG, GRKs and B-arrestins appear to have novel actions in the heart through signaling systems that are down-stream of desensitized GPCRs. GRK2 is the most abundant GRK isoform expressed in myocardium and through our close collaboration with Drs. Rockman and Lefkowitz over the last 15 years, we have uncovered a clear importance of GRK2 in the failing heart via its regulatory control on p-adrenergic receptor (PAR) signaling. Recent data by us within this PPG has elucidated that GRK2 may have influences on cardiac signaling and function not only through regulation of BAR-mediated contractile function but also through unique cellular targets, including cell survival pathways altering progression of chronic heart failure (HF). Consistent with this, data will be presented in this proposal demonstrating that GRK2 can increase myocardial apoptosis after ischemic injury. Moreover, we present data showing that GRK2 is present in mitochondria and has a unique relationship with nitric oxide synthase (NOS) and nitric oxide (NO). This includes the collaborative finding with members of this PPG group showing, for the first time, that GRK2 can be S-nitrosylated. It appears that NO bioavailability regulates GRK2's activity towards BARs. Therefore, through novel protein-protein interactions or through unique cellular localization and regulation, GRK2 appears to have multi-functions critical for normal cardiac function and adaptation during cardiac stress, especially ischemic injury, which can lead to HF. In this Project, novel aspects of GRK2 in the heart will be explored, which is inter-related and complemented by the other projects focused on myocyte PAR signaling, its regulation by other GRKs and B-arrestin function and regulation. This Project will also be greatly aided by the scientific Cores within this PPG. Our Central Hypothesis is that GRK2 plays important roles in the signaling and physiology of the heart that becomes critical in injured and compromised myocardium, especially after ischemia. This includes effects beyond padrenergic dependent contractile function and involves novel GRK regulation, cellular localization and downstream targets. The Specific Aims are: (1) To determine the mechanistic role of GRK2 in the pathophysiology of myocardial ischemic injury including myocyte survival;(2) To investigate the in vivo significance of a GRK2 - nitric oxide synthase (NOS) and NO linkage and mechanistic relationship in the heart;and (3) To determine the physiological role of S-nitrosylation and its regulation of GRK2 and its activity in the heart.